琼脂糖凝胶基片用于蛋白质与抗原分子的固定研究与表征

Agarose-gel substrate used for protein and antige immobilization and characterization

目的探索一种新型的适用于生物分子偶联的固相载体,并对其用于蛋白质与抗原分子微阵列制作的实验条件和检测结果进行评价。方法于氨基硅烷化的载玻片表面制作琼脂糖凝胶膜,经NaIO4氧化和戊二醛分子偶联后,得到3种不同的凝胶表面,分别考察其固定生物分子的偶联效率、检测敏感度和稳定性能,并用原子力显微术和X射线光电子能谱对其表面进行表征和对比研究。结果未经氧化的琼脂糖凝胶基片固定生物分子的能力和检测敏感度低于经NaIO4氧化与后续醛化处理的基片,其中又以后者的应用效果最佳,温度因素(实验温度分别为37℃、25℃和4℃)对用此法制作的蛋白质与抗原分子微阵列的使用无显著影响。表征结果提供了琼脂糖凝胶表面固定生物分子的直接证据和内在机制。结论经修饰处理的琼脂糖凝胶基片可有效固定不同的生物分子,用其作为固相表面制作的蛋白质与抗原分子微阵列可取得较为理想的实验效果。

Objective To investigate a novel solid support, which is suitable for biomolecules cross llnking,and evaluate the experimental conditions of protein and antigen mieroarray preparation as well as assay results by which detection.Methods Agarose-gel layer was prepared on the surface of APTES-modified slides, followed by oxidation with NaIO4 and cross linking with glutaraldehyde （GA）. Finally, 3 different agarose-gel substrates were achieved. Further more, proteins which atteched on these supports were reviewed,such as the effciency of HRP immobilization,the sensitivity of solid-phase molecules detection and the stability of mieroarray in employment. Atomic force microscopy（AFM）and X-ray photoelectron spectroscopy （XPS）techniques were also utilized to characterize the agarose-gel surface. Results The immobilization efficieney of HRP and detection sensitivity of biomolecules on none oxidated agaroso-gel Surface were lower than that on which oxidated by NaIO4 and subsequently modified with GA. Among them,the latter which applied in practice was optimal.No significant influence of temperature complications（ 37℃ ,25℃ and 4℃,respectively）observed in the use of this protein and antigen mieroarray. Characterization result provided a visible and internal mechanism evidence for biomolecules immobilized on agarose-gel substrate. Conclusion Different biomolecules could be firmly immobilized on the surface which oxidized and chemically modified substrate, the satisfactory results could be achieved in the use of this protein and antigen microarrays.